The objectives of this project, and related progress in the past year (2013-2014), are reviewed below. (1) DEVELOPMENT OF A REVERSE GENETICS SYSTEM THAT CAN BE USED TO MODIFY RV ANTIGENICITY AND VIRULENCE. Recently, we developed an RV single-segment reverse genetics system that allows replacement of the segment 8 (s8) RNA of the temperature-sensitive mutant virus, tsE, with a recombinant s8 RNA that encodes a fully functional NSP2. Using this system, we were able to engineer recombinant (r)RVs with s8 RNAs that were longer than wildtype, due to the introduction of sequence duplications and heterologous sequences. Subsequently, we have pursued two lines of experiments designed to generate rRVs with s8 RNAs that express foreign protein. (1) T7 transcription vectors for s8 were constructed in which a translational 2A skip element linked to a Gaussia luciferase ORF was inserted into the NSP2 ORF. Reverse genetics experiments to recover rRVs with the modified s8 RNA were attempted using a cell line (MA104-NSP2R) that expresses wildtype NSP2 and an shRNA targeting the tsE s8 RNA. (2) T7 transcription vectors for s8 were constructed in which a ribosomal-readthrough element derived from Colorado Tick Fever Virus (CTFV) segment 9 linked to a HA-ubiquitin ORF was inserted immediately downstream of an intact NSP2 ORF. Reverse genetics experiments to recover rRVs with the modified s8 RNA were attempted using a cell line expressing an shRNA that targeted the tsE s8 RNA. Analysis of virus in cell lysates obtained from reverse genetics experiments suggest that rRVs with the engineered s8 RNAs may be present, but at levels too low to be recoverable. Currently, we are modifying reverse genetics procedures to improve the efficiency of rRV formation and recovery. Generation of rRVs that express foreign proteins may be useful for engineering next generation RV vaccines that provide protection not only against RV disease, but also against diseases caused by other enteric pathogens. (2) ELUCIDATION OF RV MECHANISMS THAT SUBVERT HOST ANTIVIRAL PATHWAYS. The segmented RV genome allows the generation of new virus strains through reassortment. However, the vast majority of human RVs have either of two genotype constellations: G1/3/4/9/12-P8-I1-R1-C1-M1-A1-N1-T1-E1-H1 (genogroup 1) or G2-P4-I2-R2-C2-M2-A2-N2-T2-E2-H2 (genogroup 2). Thus, human RVs appear to be under strong selective pressures that favor the maintenance of certain genotype constellations, despite their opportunity to reassort with large numbers of co-circulating genetically-distinct human and animal RVs. The nature of the barriers that select against the emergence of reassortant strains is not understood, but may reflect the co-evolution of RV RNAs and/or proteins that function optimally when maintained as sets. In addition, adaptation of RVs to select hosts may have given rise to viral proteins that are species specific in their function. These latter proteins likely include the RV nonstructural protein, NSP1, which we have shown is an antagonist of host innate immune responses. NSP1 is a putative viral E3 ubiquitin ligase containing a conserved N-terminal RING domain and a highly variable C-terminal targeting domain. By phylogenetic analysis, we determined that the 18 NSP1 genotypes described to date can be resolved into 3 groups: OSU-like, UK-like, and SA11-4F-like. The OSU-like NSP1 group includes only the A1, A2, and A8 genotypes, and thus nearly all human RVs and select porcine RVs belong to this group. OSU-like NSP1 proteins are characterized by the ability to prevent the function of beta-TrCP, a cellular protein critical to NFkB activation and to the induction of host antiviral responses. As we discovered, a hallmark feature of OSU-like NSP1 proteins is the presence of a phosphodegron-like motif (DSGxS) in the C-terminal targeting domain; this motif mediates the interaction of NSP1 with beta-TrCP. RVs with UK-like and SA11-4F-like NSP1 proteins are almost exclusively animal strains; their NSP1 proteins target interferon regulatory factors (IRF3, IRF5, and/or IRF7), instead of beta-TrCP, and lack phosphodegron-like motifs. These findings indicate that the NSP1 proteins of human/porcine RVs (OSU-like) are functionally distinct from the NSP1 proteins of most animal RVs (UK and SA11-4F-like). Hence, the emergence of human reassortant RVs expressing animal NSP1 proteins as significant causes of human disease seems improbable, given that such reassortants would lack NSP1 activities normally associated with human RVs and that likely influence virulence and viral pathogenesis. (3) ANALYSIS OF THE DIVERSITY AND EVOLUTION OF THE RV GENOME. (a) RVs with G10P11 genotype specificity have been associated with symptomatic and asymptomatic neonatal infections in Vellore, India. To identify possible viral genetic determinants that affect RV pathogenesis, the genome sequences of G10P11 RVs in stool samples of 19 neonates with symptomatic infections and 20 neonates with asymptomatic infections were determined by Sanger and next-generation sequencing. The data showed that all 39 viruses had identical genotype constellations (G10-P11-I2-R2-C2-M2-A1-N1-T1-E2-H3), the same as the previously characterized symptomatic Vellore isolate N155. The data also showed that the RNA and deduced protein sequences of all the Vellore G10P11 viruses were nearly identical; no nucleotide or amino acid differences were found that correlated with symptomatic versus asymptomatic infection. Next-generation sequencing data revealed that some stool samples, both from neonates with symptomatic and asymptomatic infections, also contained one or more positive-strand RNA viruses (Aichi virus, astrovirus, or salivirus/klassevirus) suspected of being potential causes of pediatric gastroenteritis. However, none of the positive-strand RNA viruses could be causally associated with the development of symptoms. These results indicate that the diversity of clinical symptoms in Vellore neonates does not result from genetic differences among G10P11 RVs; instead, other undefined factors appear to influence whether neonates develop gastrointestinal disease symptoms. (b) Genome reassortment allows RV to acquire advantageous genes and adapt in the face of selective pressures. Yet, reassortment may also impose fitness costs if it unlinks genes/proteins that have accumulated compensatory, co-adaptive mutations and operate best when kept together. To better understand human RV evolutionary dynamics, we analyzed the genome sequences of 135 strains (genotype G1/G3/G4-P8-I1-C1-R1-A1-N1-T1-E1-H1) that were collected at a single location in Washington, DC, during the years of 1974-1991. Intra-genotypic phylogenetic trees were constructed for each viral gene using the nucleotide sequences, thereby defining novel allele-level gene constellations (GCs) and illuminating putative reassortment events. The results showed that RVs with distinct GCs co-circulated during the vast majority of the collection years, and that some of these GCs persisted in the community, unchanged by reassortment. To investigate the influence of protein co-adaptation on GC maintenance, we performed a mutual information-based analysis of the concatenated amino acid sequences and identified an extensive co-variance network. Unexpectedly, amino acid co-variation was highest between VP4 and VP2, which are structural components of the RV virion that are not thought to directly interact. These results suggest that GCs may be influenced by the selective constraints placed on functionally co-adapted, albeit non-interacting, viral proteins. This work raises important questions about the mutation-reassortment interplay and its impact on human RV evolution.